Abstract
The Protein Data Bank in Europe (PDBe), a founding member of the Worldwide Protein Data Bank (wwPDB), actively participates in the deposition, curation, validation, archiving and ...dissemination of macromolecular structure data. PDBe supports diverse research communities in their use of macromolecular structures by enriching the PDB data and by providing advanced tools and services for effective data access, visualization and analysis. This paper details the enrichment of data at PDBe, including mapping of RNA structures to Rfam, and identification of molecules that act as cofactors. PDBe has developed an advanced search facility with ∼100 data categories and sequence searches. New features have been included in the LiteMol viewer at PDBe, with updated visualization of carbohydrates and nucleic acids. Small molecules are now mapped more extensively to external databases and their visual representation has been enhanced. These advances help users to more easily find and interpret macromolecular structure data in order to solve scientific problems.
The Worldwide PDB recently launched a deposition, biocuration, and validation tool: OneDep. At various stages of OneDep data processing, validation reports for three-dimensional structures of ...biological macromolecules are produced. These reports are based on recommendations of expert task forces representing crystallography, nuclear magnetic resonance, and cryoelectron microscopy communities. The reports provide useful metrics with which depositors can evaluate the quality of the experimental data, the structural model, and the fit between them. The validation module is also available as a stand-alone web server and as a programmatically accessible web service. A growing number of journals require the official wwPDB validation reports (produced at biocuration) to accompany manuscripts describing macromolecular structures. Upon public release of the structure, the validation report becomes part of the public PDB archive. Geometric quality scores for proteins in the PDB archive have improved over the past decade.
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•Validation reports are available for X-ray, NMR, and EM structures in the PDB•Preliminary reports obtained at deposition, stand-alone servers, and programmatically•Official reports from biocuration should be submitted with manuscripts•Quality metrics for protein structures have improved over the past decade
Gore et al. describe the community-recommended validation reports, produced by wwPDB at deposition and biocuration of PDB submissions, and integrated into the archive of publicly released PDB entries. The authors also show that the quality of protein structures has improved over the last decade.
CORM-3, fac-Ru(CO)3Cl(κ2-H2NCH2CO2), is a well-known carbon monoxide releasing molecule (CORM) capable of delivering CO in vivo. Herein we show for the first time that the interactions of CORM-3 with ...proteins result in the loss of a chloride ion, glycinate, and one CO ligand. The rapid formation of stable adducts between the protein and the remaining cis-RuII(CO)2 fragments was confirmed by Inductively Coupled Plasma-Atomic Emission Spectrocopy (ICP-AES), Liquid-Chromatography Mass Spectrometry (LC-MS), Infrared Spectroscopy (IR), and X-ray crystallography. Three Ru coordination sites are observed in the structure of hen egg white lysozyme crystals soaked with CORM-3. The site with highest Ru occupancy (80%) shows a fac-(His15)Ru(CO)2(H2O)3 structure.
A few ruthenium based metal carbonyl complexes, e.g. CORM-2 and CORM-3, have therapeutic activity attributed to their ability to deliver CO to biological targets. In this work, a series of related ...complexes with the formula Ru(CO)3Cl2L (L = DMSO (3), L-H3CSO(CH2)2CH(NH2)CO2H) (6a); D,L-H3CSO(CH2)2CH(NH2)CO2H (6b); 3-NC5H4(CH2)2SO3Na (7); 4-NC5H4(CH2)2SO3Na (8); PTA (9); DAPTA (10); H3CS(CH2)2CH(OH)CO2H (11); CNCMe2CO2Me (12); CNCMeEtCO2Me (13); CN(c-C3H4)CO2Et) (14)) were designed, synthesized and studied. The effects of L on their stability, CO release profile, cytotoxicity and anti-inflammatory properties are described. The stability in aqueous solution depends on the nature of L as shown using HPLC and LC-MS studies. The isocyanide derivatives are the least stable complexes, and the S-bound methionine oxide derivative is the more stable one. The complexes do not release CO gas to the headspace, but release CO2 instead. X-ray diffraction of crystals of the model protein Hen Egg White Lysozyme soaked with 6b (4UWN) and 8 (4UWN) shows the addition of Ru(II)(CO)(H2O)4 at the His15 binding site. Soakings with 7(4UWN) produced the metallacarboxylate Ru(COOH)(CO)(H2O)3(+) bound to the His15 site. The aqueous chemistry of these complexes is governed by the water-gas shift reaction initiated with the nucleophilic attack of HO(-) on coordinated CO. DFT calculations show this addition to be essentially barrierless. The complexes have low cytotoxicity and low hemolytic indices. Following i.v. administration of CORM-3, the in vivo bio-distribution of CO differs from that obtained with CO inhalation or with heme oxygenase stimulation. A mechanism for CO transport and delivery from these complexes is proposed.
The complex fac-Mo(CO)(3)(histidinate)Na has been reported to be an effective CO-Releasing Molecule in vivo, eliciting therapeutic effects in several animal models of disease. The CO releasing ...profile of this complex in different settings both in vitro and in vivo reveals that the compound can readily liberate all of its three CO equivalents under biological conditions. The compound has low toxicity and cytotoxicity and is not hemolytic. CO release is accompanied by a decrease in arterial blood pressure following administration in vivo. We studied its behavior in solution and upon the interaction with proteins. Reactive oxygen species (ROS) generation upon exposure to air and polyoxomolybdate formation in soaks with lysozyme crystals were observed as processes ensuing from the decomposition of the complex and the release of CO.
We have performed experiments on the crystallization of two low molecular weight, positively charged proteins, lysozyme and ribonuclease A, using ionic liquids as either crystallization additives or, ...in particular cases, as precipitating agents. The ionic liquids (ILs) have been ordered according to their salting-in/out ability and the relative position of these ionic liquids in this ranking has been rationalized by considering their hydration properties (positive-negative, hydrophobic-hydrophilic). The ability to screen the effective charge of cationic proteins and aid protein nucleation (salting-out) has been shown to be superior for large polarizable anions with low charge density, negatively hydrated-Cl super(-), Br super(-), SCN super(-), methane-C sub(1)SO sub(3) super(-) and ethanesulfonates C sub(2)SO sub(3) super(-), than for anions with a relatively stable hydration shell, positively hydrated-lactate Lac super(-), butylsulfonate C sub(4)SO sub(3) super(-) and acetate Ac super(-). Upon increasing the background salt concentration, where electrostatic interactions are already effectively screened, the ability of the IL ions to stabilize proteins in solution (salting-in) has been shown to increase as the ions are likely to migrate to the non-polar protein surface and lower protein-water interfacial tension. This tendency is enhanced as the focus moves from those ions with positively hydrated hydrophilic compartments (e.g.Ac super(-)) to those with negatively hydrated groups (e.g.C sub(1)SO sub(3) super(-)) and the prevailing hydrophobic hydration (e.g.C sub(4)SO sub(3) super(-)). The observed inversion in the relative effect of ILs on protein crystallization with increasing ionic strength of the aqueous media has been interpreted as the differing effects of ion adsorption: charge screening and interfacial tension modification. Moreover, this work can further help in our understanding of the influence of ionic liquids on conformational changes of biomacromolecules in solution. Identification of the specific incorporation sites for choline and acetate ions, localized in two lysozyme crystals grown in pure IL solutions without any buffer or inorganic precipitant, can give us some insight into the role of the ionic liquid ions in protein structure development.
Nitrate reductase from
Desulfovibrio desulfuricans
ATCC 27774 (DdNapA) is a monomeric protein of 80 kDa harboring a bis(molybdopterin guanine dinucleotide) active site and a 4Fe–4S cluster. Previous ...electron paramagnetic resonance (EPR) studies in both catalytic and inhibiting conditions showed that the molybdenum center has high coordination flexibility when reacted with reducing agents, substrates or inhibitors. As-prepared DdNapA samples, as well as those reacted with substrates and inhibitors, were crystallized and the corresponding structures were solved at resolutions ranging from 1.99 to 2.45 Å. The good quality of the diffraction data allowed us to perform a detailed structural study of the active site and, on that basis, the sixth molybdenum ligand, originally proposed to be an OH/OH
2
ligand, was assigned as a sulfur atom after refinement and analysis of the
B
factors of all the structures. This unexpected result was confirmed by a single-wavelength anomalous diffraction experiment below the iron edge (
λ
= 1.77 Å) of the as-purified enzyme. Furthermore, for six of the seven datasets, the S–S distance between the sulfur ligand and the S
γ
atom of the molybdenum ligand Cys
A140
was substantially shorter than the van der Waals contact distance and varies between 2.2 and 2.85 Å, indicating a partial disulfide bond. Preliminary EPR studies under catalytic conditions showed an EPR signal designated as a turnover signal (
g
values 1.999, 1.990, 1.982) showing hyperfine structure originating from a nucleus of unknown nature. Spectropotentiometric studies show that reduced methyl viologen, the electron donor used in the catalytic reaction, does not interact directly with the redox cofactors. The turnover signal can be obtained only in the presence of the reaction substrates. With use of the optimized conditions determined by spectropotentiometric titration, the turnover signal was developed with
15
N-labeled nitrate and in D
2
O-exchanged DdNapA samples. These studies indicate that this signal is not associated with a Mo(V)–nitrate adduct and that the hyperfine structure originates from two equivalent solvent-exchangeable protons. The new coordination sphere of molybdenum proposed on the basis of our studies led us to revise the currently accepted reaction mechanism for periplasmic nitrate reductases. Proposals for a new mechanism are discussed taking into account a molybdenum and ligand-based redox chemistry, rather than the currently accepted redox chemistry based solely on the molybdenum atom.
Cernumidine and isocernumidine were identified in the ethanol extract of the leaves of Solanum cernuum Vell. together with four known phenolic compounds. The alkaloids have a natural ...(2-aminopyrrolidin-1-yl)carboxamidine alkaloidal base acylated with isoferulic (3-hydroxy-4-methoxycinnamic) acid with Z and E configurations, respectively. The structures were elucidated on the basis of 1D- and 2D-NMR data and the structure of cernumidine was confirmed by X-ray analysis. Cernumidine displayed inhibition of interleukin-8 production by HT-29 colon carcinoma cells. This fact could orient further research in gastric cancer prevention and treatment.
Ligand-based methods play a crucial role in virtual screening when the 3D structure of the target is not available. This study discusses the results of a validation study of the CSD field-based ...ligand screener using a novel benchmarking data set containing 56 targets. The data set was created starting from the target UniProt IDs in a previously published data set (i.e., the AZ data set), by mining ChEMBL to find known active molecules for these targets and by using DUD-E to generate property-matched decoys of the identified actives. Several experiments were performed to assess the virtual screening performance of the new method. One of its strengths is that it can use an overlay of multiple flexible ligands as a query without the need to run several parallel calculations with one ligand at a time. Here, we discuss how changes to different parameter settings or adoption of different query models can influence the final performance compared to the performance when using the experimentally observed overlay of ligands. We have also generated the enrichment scores based on three external benchmark data sets to enable the comparison with existing methods previously validated using these data sets. Here, we present results for the standard DUD-E data set, the DUD-E+ data set, as well as the DUD_Lib_VS_1.0 data set which was designed for ligand-based virtual screening validation and hence is more suitable for this type of methods.
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